The emergence of antibiotic-resistant bacteria has become a critical health concern; hence, the design of new agents which will combat the problem of developing resistance to antibiotics is of major interest. The antibiotic novobiocin is produced by Streptomyces spheroides and is a part of the aminocoumarin family of antibiotics whose mode of action is the inhibition of the type II DNA topoisomerase DNA gyrase. The structure of novobiocin consists of three moieties: a noviose sugar, a 3-substituted coumarin ring and a prenylated 4-hydroxybenzoic acid moiety. It is suggested that the noviose sugar moiety is important for recognition of the target gyrB; therefore, it is of great interest to study the enzymes involved in the biosynthesis and subsequent reactions of the noviose moiety of novobiocin. The focus of this research proprosal is divided into three specific aims: 1) characterization and substrate specificity study of NovM, the putative glycosyltransferase for glycosylation of the novobiocic acid by TDP-noviose 2) characterization and specificity studies of NovP and NovN, the noviose tailoring enzymes in the biosynthesis of novobiocin and 3) the in vitro reconstitution of TDP-noviose biosynthesis. The genes encoding NovT,U,W,S,M,P and N will be cloned from genomic DNA isolated from Streptomyces spheroides, the proteins expressed in E.coli and purified by Ni (II)-affinity chromatography. Characterization of enzyme activity in each case will be accomplished using established assays. NovM, P and N will be assayed for activity with normal and modified mbstrates using established HPLC assays. It is anticipated that these studies will advance the development of biosynthetic combinatorial libraries of sugar-modified novobiocin analogs. [unreadable] [unreadable]